Serum-free mammalian cell culture medium, and uses thereof

Abstract

The present invention provides a cell culture medium formulation that supports the in vitro cultivation, particularly in suspension, of mammalian cells, particularly epithelial cells and fibroblast cells, and methods for cultivating mammalian cells in suspension in vitro using these media. The media comprise a basal medium and a polyanionic or polyanionic compound, preferably a polysulfonated or polysulfated compound, and more preferably dextran sulfate. The present invention also provides chemically defined, protein-free eukaryotic cell culture media comprising an iron chelate and zinc, which is capable of supporting the growth (and particularly the high-density growth of mammalian cells) in suspension culture, increasing the level of expression of recombinant protein in cultured cells, and/or increasing virus production in cultured cells.

What is claimed is: 
     
       1. A suspension cell culture comprising a serum-free and protein-free cell culture medium and a Chinese Hamster Ovary (CHO) cell, wherein said culture medium comprises a transferrin substitute and an insulin substitute, said transferrin substitute comprising an iron or an iron containing compound and said insulin substitute comprising a zinc or a zinc containing compound, wherein said culture medium supports increased growth of the CHO cell in suspension as compared to cultivation in FMX-8 medium without supplementation of serum, and without supplementation of protein, and without expression of an exogenous recombinant protein in the CHO cell. 
     
     
       2. The suspension cell culture of  claim 1 , wherein said CHO cell expresses a recombinant protein or a virus. 
     
     
       3. The suspension cell culture of  claim 2 , wherein said recombinant protein is a secreted protein. 
     
     
       4. The suspension cell culture of  claim 2 , wherein said recombinant protein is harvested from the used cell culture medium. 
     
     
       5. The suspension cell culture of  claim 1 , wherein said cell is cultured in a bioreactor. 
     
     
       6. The suspension cell culture of  claim 1 , wherein said increased growth produces higher concentrations of recombinant protein. 
     
     
       7. The suspension cell culture of  claim 1 , wherein said medium further comprises glucose at a final concentration of about 1.000 g/L to about 12.60 g/L. 
     
     
       8. The suspension cell culture of  claim 1 , wherein said medium comprises a trace element selected from the group consisting of barium, potassium, cobalt, manganese, chromium, copper, nickel, molybdenum, fluorine, silver, rubidium, tin, zirconium, cadmium, aluminum, germanium, titanium, vanadium, silicon and magnesium. 
     
     
       9. The suspension cell culture of  claim 1 , wherein said medium comprises a total amino acid concentration of about 1.404 g/L to about 12.42 g/L. 
     
     
       10. The suspension cell culture of  claim 1 , wherein said increased growth is about 1.5×10 6  to 2×10 7  cells/ml. 
     
     
       11. The suspension cell culture of  claim 1 , wherein the medium further comprises one or more ingredients selected from the group consisting of L-arginine, L-asparagine.H2O, L-aspartic acid, L-glutamic acid, L-histidine, hydroxyl-L-proline, L-isoleucine, L-leucine, L-lysine.HCl, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cystine.2HCl, Na 2 HPO 4 , pyridoxine.HCl, thiamine.HCl, glutathione, cupric sulfate.7H 2 O, cadmium chloride.5H 2 O, cobalt chloride.2H 2 O, stannous chloride.2H 2 O, manganous sulfate.H 2 O, nickel sulfate.6H 2 O, sodium metavanadate, ammonium molybdate.4H 2 O, barium acetate, potassium bromide, potassium iodide, chromium sulfate, sodium fluoride, silver nitrate, rubidium chloride, zirconyl chloride, aluminium chloride, germanium dioxide, titanium tetrachloride, sodium metasilicate, magnesium chloride (anhydrous), D-calcium pantothenate, calcium nitrate.4H 2 O, potassium chloride, ascorbic acid magnesium salt phosphate, pluronic F68 10% solution, Na 2 HPO 4 , D-glucose, folic acid, riboflavin, biotin, choline chloride, niacinamide, inositol, sodium pyruvate, vitamin B-12, β-mercaptoethanol, para-amino benzoic acid, β-glycerophosphate, sodium selenite, ethanolamine.HCl, spermine, putrescine.2HCl, monothioglycerol, and sodium bicarbonate, wherein each of said ingredients is present in an amount which supports the high-density growth of Chinese hamster ovary cells in suspension culture and/or the expression of recombinant protein. 
     
     
       12. The suspension cell culture of  claim 2 , wherein said recombinant protein or said virus is isolated.